Many human disease states are identified on the basis of immunoassay techniques which rely upon the specificity between immunoglobulin, whether monoclonal or polyclonal, and their respective binding partners, which may be haptens, antigens, or other analytes, all of which may hereafter be collectively and interchangeably referred to herein as "ligands" and "ligand binding partners." Furthermore, "ligand" also means any molecule having an affinity to bind or complex with a "ligand binding partner", including chelators, immunobinders, nucleic acid strands, bioreceptors, and hydrophobic binders. Over the past fifteen or so years, there has been a substantial amount of effort involved in the development of immunoassay techniques utilizing the so-called sandwich and competitive techniques. The sandwich technique involves the immobilization of an antigen by one antibody and then subsequent labeling by attachment of a second antibody having associated therewith a detectable label. Reverse immunoassays for the detection of antibody are similar but instead put antigen on the surface for reaction with the sample antibody. Competitive techniques are useful for antigens having only a single epitopic site for reaction with an antibody. Accordingly, and as the name implies, such techniques rely upon the competition of the antigen with another labeled antigen for a binding site on an immobilized antibody. The substitutions necessary for antibody detection tests are obvious and need not be covered here in any great detail.
Of great importance in the laboratory is the development of highly sensitive techniques which can be run in either batch random access, panel, or stat modes. Preferably, such techniques will be homogeneous in nature, i.e., and as used herein, they will be conducted solely within one container without any accompanying requirement to physically separate out components following reactions during the assay.
It is one object of the present invention to provide a new immunoassay system which is highly sensitive and which is homogeneous in nature.
U.S. Pat. 3,939,350 to Kronick and the Kronick citations therein referenced describe an immunoassay system which allows for the measurement of biochemical analytes by fluorescence in a liquid sample. Kronick employs a physical phenomenon known under the name of total internal reflectance. This optical phenomenon occurs wherein light, when directed through a high refractive index material toward the interface of that material with a second material having a lower refractive index at greater than a critical angle, all light is reflected from that interface save for a microscopic evanescent wave which propagates into the second material for only a short distance. The second material may, for instance, be water or another aqueous medium in which an assay is being conducted. Kronick noted that when he brought materials which had been fluorescently labeled down to the interface and within the field of the evanescent wave, he could energize the fluorescent molecules and detect fluorescence which then emanated into the overlying solution. The Kronick system, however, looks at fluorescence which cannot be readily modified by alteration of the fluorescent labels in order to suit the system under study. Due to the nature of the specificity of the fluorescent label with respect to the wavelength of the excitation frequency, one is limited to a discrete light source providing the critical excitation frequency. To date, most investigators favor the He-Ne laser light source due to its reliability and low cost as well as the low cost of associated optics. Such a light source, however, brings concomitant difficulties in tailoring fluorescent molecules to be excited by the He-Ne laser output. The organic, inorganic, and bio-organic techniques required are especially difficult to control in the immunoassay arena. Further, Kronick's reliance on fluorescence is accompanied by additional disadvantages associated with bleaching of the fluorescent molecules and generally critical matching of fluorescent molecule excitation wavelength with laser output wavelength necessary to obtain good quantum efficiency.
It is an object of the present invention to provide a new immunoassay system which avoids the disadvantages associated with fluorescent labels and the criticality associated with matching an excitation source.
It is another object of the present invention to employ the principles of total internal reflection but with far greater flexibility regarding the choice of illumination sources. U.S. Pat. 4,181,441 to Noller describes a system similar to that of Kronick. Noller, however, taught that the assay should be conducted by measurement of light absorption in a liquid sample which could then be correlated to the presence of biochemical analytes. Although the Noller system employs different physical principles than the Kronick system, light absorption measurements are similarly subject to poorer signal-to-noise ratios due to small differences in large light signals thereby making such a system inherently less sensitive than desired. It is another object of the present invention to avoid employing light absorption measurements while still gaining the advantages to be provided by the total internal reflectance phenomenon.
U.S. Pat. No. 4,521,522 to Lundstrom teaches yet another immunoassay based upon reflectance and the use of Brewster s angle. This system relies upon a different optical phenomenon wherein directing a light beam, polarized in the plane of incidence, upon an interface, for example that formed between plastic and liquid, results in the transmission of a strong light beam into the liquid when such light strikes the interface at the Brewster angle. At the Brewster angle, substantially no light is reflected.
The Brewster angle is a function of the refractive indices of the two materials as well as the direction of polarization. Lundstrom noted that upon the growth of a biochemical layer at the interface, the Brewster angle condition would be disrupted resulting in increasing light reflectance, particularly at angles less than the Brewster angle. Unfortunately, the Lundstrom assay only works effictively with a wash step since the transmission of the beam into the liquid also results in the generation of light scatter and thus a spurious signal.
It is another object of the present invention to utilize light scatter but to avoid light scatter generated by the transmission of light into the liquid which occurs naturally when light is directed at an interface at the Brewster angle. Accordingly, it is yet another object of the present invention to avoid employing a Brewster angle condition.